From: John Philo <jphilo@mailway.com>
  To  : E.braswell <BRASS@UCONNVM.UCONN.EDU>
  Date: Fri, 28 Apr 2000 09:28:14 -0700

RE: incompetency; PEG

I did not mean to imply that multiple long-lived or irreversible
conformations or 'misfolds' don't exist, and indeed even an initially pure
conformation state may accumulate other partially unfolded species due to
stresses such as shear, freeze-thaw, or surface denaturation. However, I'm
not sure that this situation of a range of conformations with intermediate
binding affinities is very common today, although it may have been more so
in the early days of recombinant proteins. (I admit though my viewpoint may
be biased by the good fortune of working with people who are REALLY good at
making recombinant proteins.)

Further, I think that when you do have some damaged or misfolded species
this model of simply modulating the interaction strength may be too
simplistic. Generally such states are 'sticky' and I think it likely that
such species are going to give rise to new modes of association (probably
something we would call 'aggregation') in addition to whatever is the
'normal' association reaction. To further complicate matters, since such
species are generally pretty unstable, it is likely they may be
precipitating or otherwise changing over the course of an equilibrium
experiment.

If you do have a spectrum of interaction strengths it should definitely show
up in the kinetics as measured by surface plasmon resonance, and perhaps
also in titration calorimetry. Perhaps others could comment about that.

Emory is right, though---wouldn't it be lovely to have an analysis method
for sedimentation equilibrium that gives a distribution of interaction
strengths? It could revolutionize things as much as Walt Stafford's DCDT has
done for velocity.

Regarding PEGylation versus glycosylation, in my view Emory has asked the
question the wrong way around. The purpose of PEGylating protein
pharmaceuticals is often to try to simulate the many nice things that
glycosylation does for proteins. Both PEG and carbohydrate greatly increase
the hydrodynamic size, which can be critical for reducing rapid clearance of
small proteins through the kidneys. Both carbohydrate and PEG can increase
solubility and may reduce aggregation by covering up sticky sites on the
surface. Both may alter recognition by the immune system. One significant
difference is that the carbohydrates are often highly charged. Another is
that the carbohydrates may interact specifically with the surface; they also
apparently bind to unfolded states and indeed seem to enhance folding (see
for example J Biochem (Tokyo) 2000 Mar;127(3):427-33). Carbohydrates
generally increase the stability of the native state; PEG I think is more
likely to be destabilizing.

Lastly, with regard to estimating degrees of glycosylation or PEGylation, I
think Emory is referring to the idea of using the XL-I and a synthetic
boundary cell as a refractive index detector to measure the dn/dc and then
compute composition based on the dn/dc of protein versus polymer. I believe
there are two references to that general method, but using chromatography
detectors rather than the centrifuge, for PEG or carbohydrate, by Michael
Kunitani at Chiron, but I can't put my finger on the exact ones right now.
Write me if you need them and I'll dig that out.

John Philo
-----Original Message-----
From: E.braswell [BRASS@UCONNVM.UCONN.EDU]">mailto:BRASS@UCONNVM.UCONN.EDU]
Sent: Thursday, April 27, 2000 5:52 PM
To: rasmb@alpha.bbri.org
Subject: incompetency


John, I don't know much about folding, or its dynamics, but can't molecules
be locked in a number of nearly similar configurations or be "misfolded"
(as I've heard the term used by people who produce proteins by recombinant
methods)?   This was what I was thinking of, and a number of such misfolded
species could conceivably cause a range of lnKs.
It would be nice if we could produce a graph of the distribution of lnKs for
a given species in solution in order to characterize these.
I have contributed my 2 cents more on this topic in the last 2 days than I
usually do in a year--so please bear with me and give us one more John!
and any others please.
  Next question, does anyone know if glycosylation can do most of the nice
things that PEGylation does for pharmaceuticals?  Incidentally, I am
ignorant of the literature, but I have lately tried estimating degrees
of PEGylation and glycosylation, by measuring sed velocity with both
interference and absorption at 280.  I know of some gotchas but there are
probably more.  Any references anybody?
                                           Regards, Emory

Emory H. Braswell
Professor of Biophysics and Head,
National Analytical Ultracentrifugation Facility
U-149
University of Connecticut
STORRS, CT 06269-3149
TEL 860 486 5032
FAX         5005
E.BRASWELL@UCONN.EDU